Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy

Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction.


Results
Mitophagy is inhibited during loss of PHB1. Phb1 iΔIEC mice exhibit loss of intestinal epithelial PHB1 expression (Fig. S1A) and manifest spontaneous ileal inflammation driven by mitochondrial dysfunction in IECs within 12 weeks after the induction of Phb1 deletion 29 . Paneth cell defects, such as altered lysozyme granule staining (diffuse, disordered, and depleted), decreased electron-dense secretory granules, and vesiculated ER, were evident prior to inflammation as early as 1 week after the induction of Phb1 deletion (Figs. 1A and S1B) 29 . Since these Paneth cell defects are reminiscent of mice deficient in autophagy genes Atg16l1, Atg7, or Atg5 9 , we measured autophagosome formation by LC3II in ileal IECs of Phb1 fl/fl and Phb1 iΔIEC mice. One week after the induction of Phb1 deletion, autophagosomes were visualized as LC3II + puncta and were increased in the crypt base in Paneth cells marked by lysozyme + staining and in villi in secretory goblet cells marked by Muc2 + staining (Figs. 1B,C and S1C). This same increase in LC3II puncta has been demonstrated previously in Paneth and goblet cells of autophagy deficient mice 7,8 . Of note, goblet cells of Phb1 iΔIEC mice also exhibit abnormalities during the progression to ileitis, but are less susceptible to mitochondrial dysfunction than Paneth cells 29 . Increased autophagosomes during Phb1 deletion could result from increased autophagy activation or decreased autophagosome degradation. Since abundance of p62 is a well-established marker of autophagosome turnover, we next assayed p62 in freshly isolated ileal IECs of Phb1 iΔIEC mice. p62 protein expression was variable across ileal IECs of Phb1 iΔIEC mice with some demonstrating increased abundance (Figs. 1D and S2), suggesting inhibition of autophagosome turnover in these Phb1 iΔIEC samples. As a measure of mitochondrial turnover, abundance of inner mitochondrial membrane proteins Tim50 and CoxIV was increased in IECs of some Phb1 iΔIEC mice (Figs. 1D and S2). As a more sensitive measure of mitochondrial turnover, mitochondrial DNA:nuclear DNA ratio was increased in IECs of Phb1 iΔIEC mice compared to Phb1 fl/fl mice (Fig. 1E). These increases in mitochondrial markers were not associated with increased mitochondrial biogenesis in Phb1 iΔIEC www.nature.com/scientificreports/ ileal IECs (Fig. 1F), as compared to Opa1 expression, a regulator of mitochondrial IMM fusion that has been previously shown to be regulated by PHB1 expression 30 .
These results were corroborated in vitro using the non-transformed murine intestinal epithelial cell line, Mode-K, transfected with 2 independent siRNA constructs targeting PHB1 and treated with Bafilomycin A1 (BafA) to prevent autophagosome degradation. PHB1 knockdown induced accumulation of CoxIV and Tim50 similar to that induced by BafA alone (Figs. 2A, S3, S4). Compared to siNC (siRNA negative control) cells treated with BafA, CoxIV and Tim50 failed to further increase in siPHB1 cells treated with BafA (Figs. 2A, S3, S4), indicating decreased mitophagic flux during PHB1 knockdown (Fig. 2B). Furthermore, relative mitochondrial DNA:nuclear DNA ratio was increased in siPHB1 cells (Fig. 2C).   www.nature.com/scientificreports/ We next utilized expression of a mitochondrial-targeted fluorescent tag, mito-RFP-EGFP, to measure activation of mitophagy in Mode-K cells. Using this tag, intact mitochondria are marked by RFP-EGFP merged yellow signal under basal conditions, whereas during activation of mitophagy, mitochondria appear RFP + since EGFP is quenched and finally degraded in the lysosome (Fig. 2D) 31 . Optimal conditions to elicit mitochondrial stress prior to the induction of apoptosis or severe mitochondrial alterations such as OMM rupture was determined in Mode-K cells treated with the ETC inhibitors rotenone, oligomycin, and antimycin A from 2 to 16 h (Figs. S5 and S6). Rotenone, oligomycin, and antimycin A increased the formation of mitochondrial ROS after 2 h of treatment at a similar magnitude (Fig. S5A). Unlike antimycin A, rotenone did not stimulate cytochrome C release from mitochondria to the cytosol at 2 or 8 h of treatment ( Fig. S5B,C). Additionally, only 31% of mitochondria exhibited OMM rupture after 16 h of rotenone treatment, which was significantly less severe than oligomycin or antimycin A (Fig. S6B), as previously reported 28,32 . Therefore, rotenone treatment for 2 h was utilized to induce mitochondrial stress prior to the induction of apoptosis or OMM rupture. In Mode-K cells transfected with siNC, treatment with rotenone for 2 h activated mitophagy as indicated by increased mito-RFP-EGFP-induced red signal and decreased yellow signal (Figs. 2E,F, and S7). On the contrary, cells deficient in PHB1 showed little to no activation of mitophagy by rotenone (Figs. 2E,F, and S7). Collectively, these results suggest that mitochondria are not efficiently degraded during PHB1 deficiency in non-transformed IECs. PHB1 serves as an inducer of mitophagy during increased mitochondrial ROS independently of PHB2. PHB1 and PHB2 have both independent and dependent functions that are at least, in part, regulated by cellular localization and numerous post-translational modifications 33 . For instance, previous studies demonstrated a role of PHB2 independent of PHB1 in mitophagy activation during outer mitochondrial membrane rupture through direct binding to LC3II 28 . We therefore tested whether inhibited mitophagy during PHB1 deletion in IECs was associated with a compensatory loss in PHB2 expression. Indeed, freshly isolated ileal IECs from Phb1 iΔIEC mice exhibited a concomitant loss of PHB1 and PHB2 expression (Figs. 3A, S8, and S9) with similar results in Mode-K cells during siPhb1 knockdown (Figs. 3B and S10). This linked expression of PHB1 and PHB2 has been shown previously in other systems 20,28 . Forced overexpression (OE) of PHB2 was able to restore PHB2 levels during siPhb1b knockdown (Qiagen construct) but not during siPhb1a knockdown (Invitrogen construct) (Fig. 3B), therefore siPhb1b knockdown coupled with PHB2 exogenous expression was used for further analysis. Forced expression of PHB2 lessened the inhibition of basal mitophagy by siPhb1b in vehicletreated cells as indicated by decreased relative mtDNA accumulation (Fig. 3C). This suggests concomitant loss of   Fig. 3A,B) is partially responsible for basal mitophagy inhibition during PHB1 knockdown. Rotenone treatment for 2 h induces mitophagy in siPhb1b cells with a slight but significant decrease in relative mtDNA accumulation in rotenone-treated siPhb1b cells compared to vehicle-treated siPhb1b cells (Fig. 3C). This is likely due to remaining PHB1 and PHB2 expression since the siPhb1b construct produces incomplete knockdown of PHB1 and incomplete compensatory loss of PHB2 (as shown in Fig. 3B). Interestingly, forced overexpression of PHB2 had no effect on relative mtDNA accumulation ( Fig. 3C) or mito-RFP-EGFP expression ( Fig. S11) during siPhb1b knockdown and rotenone treatment, suggesting PHB2 cannot rescue mitophagy response to rotenone during PHB1 deficiency. We reasoned this could be due to PHB2 requiring OMM rupture to induce mitophagy activation 28 coupled with 2 h rotenone not stimulating OMM rupture in our system (Figs. S5 and S6). We therefore treated with antimycin A to induce OMM rupture (Figs. S5B and S6) and noted that forced expression of PHB2 rescued antimycin A-induced relative mtDNA accumulation (Fig. 3C) or mito-RFP-EGFP expression (Fig. S11) during siPhb1b knockdown. These results suggest that mitophagy induction without robust OMM rupture (rotenone treatment) requires PHB1 independently of PHB2.

PHB1 interacts with mitophagy receptor Nix independently of Parkin. Previous studies have
established that Parkin-dependent mitophagy requires rupture of the OMM 28,34 . However, Parkin-independent mitophagy does not require OMM rupture; Parkin-independent autophagy receptors localize to the OMM to mediate mitophagy. In fact, Nix-mediated mitophagy has been shown to be induced by mitochondrial ROS accumulation without rupture of the OMM 35 . We next determined whether PHB1 interacts with mitophagy receptors and whether Parkin was involved. The mitophagy receptor Nix interacted with GST-PHB1 in protein isolated from ileal IECs of wild-type (WT) or Parkin −/− mice and Mode-K cells (Figs. 4A, S12A, S14, and S15). There was no interaction between GST-PHB1 and other mitophagy receptors including FundC1, Bnip3, Bcl2L13, Optineurin, or NDP52 (Fig. 4A). Parkin independent interaction of PHB1 with Nix was confirmed by co-immunoprecipitation assay (Figs. 4B, S16, S17). Additionally, PHB1/Nix interaction was increased after 2 h rotenone treatment (Fig. 4B). Induction of PHB1 interaction with Nix during mitochondrial stress induced by rotenone is conserved in cell types beyond IECs including HeLa cells (Fig. S12B). Induction of mitophagy by Nix involves its transcriptional upregulation, constitutive localization to the OMM, and direct interaction with LC3II 15 . During siPHB1 knockdown in Mode-K cells, there was ample rotenoneinduced Nix transcriptional activation ( Fig. S12C) but Nix did not accumulate in mitochondria during rotenone treatment (Figs. 4C and S17). Rotenone-induced LC3II association with the mitochondrial fraction was inhibited during siPHB1 knockdown (Fig. 4C). Although Phb1 iΔIEC mice exhibited similar Nix expression in ileal IECs as Phb1 fl/fl mice (Fig. S12D), less Nix interacted with LC3II in crypts of Phb1 iΔIEC mice, despite high expression of LC3II (Fig. 4D). Together, these results suggest that the IMM protein PHB1 binds to the OMM protein Nix during mitochondrial dysfunction prior to robust OMM rupture. Additionally, PHB1 is not required for induction of Nix transcription during elevated ROS but is necessary for Nix localization to mitochondria. We nexted determined whether ROS scavengers such as N-acetyl-l-cysteine (NAC) or and mitoTEMPO (mitochondrialtargeted superoxide dismutase mimetic) inhibit PHB1 and Nix interaction. Compared to vehicle-treated control cells, NAC and mitoTEMPO did not alter basal PHB1/Nix interaction by co-immunoprecipitation (Fig. S13). This suggests that dampening of ROS levels does not regulate basal interaction of PHB1 and Nix, at least in this model using ModeK cells.

PHB1 interaction with Nix is necessary for mitophagy during increased mitochondrial ROS.
To determine binding region of PHB1 crucial for interaction with Nix, Mode-K cells were transiently transfected with full-length PHB1-GFP or PHB1-GFP deletion constructs (Fig. 5A) and treated with rotenone to increase mitochondrial ROS. By co-immunoprecipitation of exogenous PHB1-GFP, PHB1-GFP Δ156-190 deletion construct exhibited little-to-no interaction with Nix (Figs. 5B, S19, and S20). Amino acids 156-190 of PHB1 are part of the Prohibitin domain and the coiled-coil domain, both of which are important for protein-protein interactions and project into the mitochondrial inner membranous space 19,21 . Overexpression of PHB1-GFP Δ156-190 in Mode-K cells inhibited mitophagy induced by rotenone as demonstrated by sustained mitochondrial DNA:nuclear DNA ratio (Fig. 5C) and sustained Tim50 protein abundance (Figs. 5D, S21). Taken together, these results suggest that PHB1 interaction with Nix is a key inducer of mitophagy during increased mitochondrial ROS.

PHB1 co-localization with Nix is increased in Crohn's disease ileal crypts. PHB1 and NIX
co-localization in ileal crypts of Crohn's disease patients (average 27 ileal crypts/patient across 6 patients) or non-IBD control patients (average 47 ileal crypts/patient across 6 patients) was visualized and quantitated by immunofluorescent staining (Fig. 6A; arrows). PHB1 and NIX co-localization was increased in Crohn's disease crypts (Fig. 6B), a disease associated with mitochondrial stress 36 .
Phb1 mediates Nix-induced mitochondrial clearance during reticulocyte maturation. An established role of Nix in mediating mitophagy is in reticulocytes when all mitochondria are eliminated during maturation to erythrocytes 37 . To validate whether Phb1 interaction with Nix is necessary for Nix-induced mitophagy, we crossed Phb1 fl/fl mice with EpoR Cre mice to generate mice with PHB1 deficiency in cells of the erythroid lineage. 23% of mouse pups were Phb1 fl/+ , 35% were Phb1 fl/fl , 42% were Phb1 fl/+ :EpoR Cre , and 0% were Phb1 fl/fl :EpoR Cre with equal males and females produced per genotype. Since Phb1 fl/fl :EpoR Cre mice were not viable, all studies utilized Phb1 fl/+ :EpoR Cre mice that exhibited 40% loss of Phb1 mRNA and protein expression in reticulocytes (Fig. S22A,B). Phb1 fl/+ :EpoR Cre mice exhibited the same body weight gain as control littermates up to 18 weeks of age and had no overt phenotype. To induce erythropoiesis, mice where retroorbitally-bled 2 consecutive days before analysis. Phb1 fl/+ :EpoR Cre mice exhibited increased reticulocytes in whole blood  S22C) and abnormally shaped erythrocytes (Fig. S22D), suggestive of a maturation defect and similar to Nix −/− mice 37 . To stimulate reticulocyte maturation and concomitant mitochondrial clearance by mitophagy, reticulocytes were purified from whole blood by CD71 + MACS magnetic bead/column isolation and cultured for 4 days. Isolated cells were validated as reticulocytes by flow cytometry (Fig. S23A). Phb1 fl/+ :EpoR Cre reticulocytes exhibited defective mitochondrial clearance as visualized by retained mitoTracker Red staining after 4 days of culture (Fig. S23B). Collectively, these results suggest that Phb1 deficiency in reticulocytes inhibits mitochondrial clearance by mitophagy, phenocopying Nix −/− reticulocytes 37 .

Discussion
Our results demonstrate that much like PHB2, PHB1 acts as a sensor for stress localized to the IMM and matrix to induce mitophagy. Unlike PHB2, PHB1 acts independently of Parkin as a mitophagy inducer via Nix in response to increased ROS not requiring OMM rupture. We speculate that PHB1 and PHB2 serve complimentary functions in the induction of mitophagy with PHB1 acting independently of Parkin during mitochondrial stress  www.nature.com/scientificreports/ (increased ROS) and PHB2 acting in a Parkin-dependent manner as reinforcement upon OMM rupture. This dual mechanism of mitochondrial incorporation into the autophagy pathway may enhance the efficiency of removal of damaged mitochondria. Membrane rupture was shown to be a necessary event for sequestration of organelles such as lysosomes and endosomes into the autophagy degradation pathway 38,39 . In contrast, our results indicate that mitochondria can be removed by mitophagy prior to OMM rupture via Nix. Nix localizes to mitochondria and endoplasmic reticulum and is a key mediator of mitophagy in cardiomyocytes and in anucleated cells such as reticulocytes and   [40][41][42][43] . Here, we demonstrate that reticulocytes deficient in Phb1 expression phenocopy Nix −/− reticulocytes with defective mitochondrial clearance, suggesting Phb1 is a crucial regulator of Nix-induced mitophagy. Expression of Nix is controlled by the transcription factor hypoxia-inducible factor 1α (Hif1α), suggesting hypoxia is a central activator of Nix-induced mitophagy [44][45][46] . Low levels of elevated mitochondrial ROS participate in hypoxia response by regulating the stability of Hif1α, whereas high levels of mitochondrial ROS induce OMM rupture, facilitating apoptosis via release of mitochondrial proteins into the cytoplasm such as Cytochrome C 18 . In this way, PHB1 may dampen mitochondrial-mediated apoptosis via Nix-induction of mitophagy prior to severe mitochondrial damage. In the intestinal epithelium, Nix is increased in inflammatory bowel disease patients and in murine colitis in a manner dependent on mitochondrial ROS 45 . Nix −/− mice are more susceptible to chemically-or adoptive T cell transfer-induced colitis 45 , supporting an important role of Nix-induced mitophagy in the protection against intestinal inflammation. Our results in Phb1 iΔIEC mice suggest that Phb1 deletion with compensatory deficiency in PHB2 inhibit mitophagy involved in the development of Crohn's diseaselike ileitis 29 . It is well-established that goblet and Paneth cells are susceptible to autophagy inhibition 7,8 ; our results demonstrate a specific role of mitophagy in these cells and in maintaining intestinal homeostasis. PHB1 interaction with Nix may be important in cells highly susceptible to mitochondrial dysfunction, such as Paneth cells 29 , as a first line of defense to remove stressed mitochondria. In our previous publication, we demonstrated that the mitochondrial-targeting antioxidant mitoTEMPO could ameliorate ileitis in Phb1 iΔIEC mice, suggesting that mitochondrial-derived ROS contributed to the pathology of this model 29 . Given the results presented here, we speculate that accumulation of damaged mitochondria due to inefficient mitophagy in Phb1 iΔIEC mice also contribute to the pathology of this model. Future studies will test whether induction of mitophagy, such as via Urolithin A treatment 47 , can dampen ileitis severity in Phb1 iΔIEC mice. The exact mechanism(s) of how PHB1 interaction with Nix facilitates Nix mitochondrial localization or Nix binding with LC3II is not known. PHB1 exhibits chaperone activities, stabilizing mitochondrial proteins for optimal function 23,24 . PHB1 may exert similar stabilizing function for Nix in the mitochondria. Although only 11 amino acids of the Nix C-terminus localizes to the intermembrane mitochondrial space, this C-terminus has been shown to be an important regulator of mitophagy 48 . Key C-terminal amino acid residues of Nix that extend into the mitochondrial intermembranous space are responsible for proper Nix-dependent mitophagy 48 . A large C-terminal proportion of PHB1 (amino acids 28-272) reaches into the intermembrane mitochondrial space facilitating protein-protein interactions via the Prohibitin domain and coiled-coil domains [19][20][21] . Our studies suggest portions of the PHB1 C-terminus are necessary for interaction, either directly or indirectly, with Nix. Interaction of Prohibitins with the C-terminus of OMM-localized proteins, such as Bif-1, Miro1, and Miro2, has been previously reported 22,49 . During mitochondrial swelling and unfolding of the IMM, the IMM can become juxtaposed to the OMM which may further facilitate interaction between proteins residing in these membranes 50 . It has also been demonstrated that post-translational modifications of Nix regulate its pro-mitophagy activity, with increased affinity for LC3II by C-terminal phosphorylation of Nix within the LIR domain 51 , dimerization of Nix, and subsequent mitophagy initiation 48 . Future studies will determine whether interaction of Nix with PHB1 are necessary for its phosphorylation and subsequent LC3II binding and/or dimerization, providing an additional layer of mitophagy regulation.
PHBs serve multiple, diverse cellular functions including regulation of cell-cycle progression, senescence, gene transcription, apoptosis, and mitochondrial fitness 26 . Global genetic deletion of Phb1 or Phb2 results in embryonic lethality in mice, suggesting an essential role in early development 30,52,53 . Although PHB1 and PHB2 function primarily in the mitochondria, independent functions of PHB1 thus far distinct from PHB2 include inhibition of Wnt signaling, interaction and regulation of SH2 proteins (Shp1, Lyn, Syk), and interaction with Ras, C-RAF, and Akt in plasma membrane lipid rafts 33 . PHB2 has been shown to regulate ERα signaling and interact with LC3II 33 . Decreased PHB1 and PHB2 expression is associated with aging and age-associated diseases with known mitophagy impairment including Parkinson's disease and Alzheimer's disease 54,55 . Phb2 deletion specifically in forebrain neurons results in neurodegenerative disease and premature death in mice 56 . The opposite is noted in many types of cancer with increased PHB1 and PHB2 expression 57 and mitophagy serving context-dependent tumor suppressor and tumor promoter functions 58 . We previously showed that PHB1 knockdown in the colorectal cancer cell lines Caco2 and HCT-116 induced mitochondrial stress and mitophagy 59 in contrast to our current findings in non-transformed IECs. We speculate that this is due to mitochondrial and metabolic adaptations during transformation with additional mechanisms of mitophagy not dependent on PHB1. In terms of inflammation, PHB1 has been shown to be protective across numerous tissue types including the intestine, liver, heart, pancreas, lung, and brain [60][61][62] whereas this is less studied for PHB2. Given the established role of mitophagy in aging, neurodegeneration, cancer, and inflammation, the functions of PHB1 and PHB2 in mitophagy likely play a mechanistic role in these diseases. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a role of PHB1 in direct binding of Parkin-independent mitophagy induction and intestinal homeostasis. PHB1 has pleiotropic roles in mitochondrial quality control, simultaneously regulating mitochondrial function and stimulating mitophagy induced by increased mitochondrial ROS.

Methods
Mouse models. Phb1 fl/fl mice (previously described 63 ) were crossed with Villin-Cre-ER T2 mice, both on the C57Bl/6 genetic background. 8-week old male and female Phb1 fl/fl and Phb1 fl/fl :Villin-CreERT2 (referred to as Phb1 iΔIEC mice 29 ) were intraperitoneally injected with 100 μl of 10 mg/ml tamoxifen (Sigma Aldrich) as previously described 64  www.nature.com/scientificreports/ 350 μl of blood was removed by orbital bleed over 2 consecutive days from EpoR Cre :Phb1 fl/+ and Phb1 fl/+ mice. Experiments were performed with age-and gender-matched littermate mice. All mice were grouped-housed in standard cages under SPF conditions and were allowed standard chow and tap water ad libitum. All experiments were approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee and our reporting follows the recommendations in the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.

Human patient mucosal biopsy immunofluorescent staining. The study design and conduct
were authorized by the Baylor University Medical Center, Dallas, Texas IRB committees (IRB 016-083) and complied with all relevant regulations and guidelines regarding the use of human study participants. The study was conducted in accordance with the criteria set by the Declaration of Helsinki. Informed consent was obtained from all subjects. Crohn's disease patients and non-IBD controls were recruited from Baylor University Medical Center during endoscopy procedure to obtain mucosal biopsies from the terminal ileum. Non-IBD controls underwent an elective endoscopy for clinical purposes, did not have a history of IBD, and lacked endoscopic and histological inflammation. Biopsies were immediately formalin-fixed and later embedded in paraffin. Immunofluorescent staining was performed as described below. Sections were visualized using Zeiss Axioskop Plus microscope. Co-localization of PHB1 and NIX was analyzed by Pearson's correlation coefficient using Fiji (ImageJ). Briefly, immunofluorescent images were converted to 8-or 16-bit, region of interest selected in one of the channels, and chose "Coloc2" plugin from "Colocalization" under "Analyze" to find the value for the Pearson's correlation coefficient.

Isolation of intestinal epithelial cells (IECs). IEC isolation was performed as previously described 66 .
Transmission electron microscopy. Distal ileum or Mode-K cells were fixed in 2% gluteraldehyde in 1× PBS, dehydrated and embedded in epoxy resin for electron microscopy. Ultrathin 70 nm sections were examined on a transmission electron microscope (Hitachi BioMedical TEM). Immunofluorescent staining. 7 μM paraffin-embedded sections of mouse or human ileum were dehydrated in xylene and ethyl alcohol gradient, incubated in 10 mM sodium citrate for antigen epitope retrieval, blocked in 5% normal donkey serum, and exposed to the following primary antibodies at 4 °C overnight: www.nature.com/scientificreports/ Immunohistochemical staining. The following antibodies were used for immunostaining: PHB1 (70R-5543, polyclonal, Fitzgerald), 7 μM paraffin-embedded sections of ileum were dehydrated in xylene and ethyl alcohol gradient, incubated in 0.3% H 2 O 2 for 30 min, washed, incubated in 10 mM sodium citrate for antigen epitope retrieval, blocked in 5% normal goat serum, and exposed to PHB1 antibody (70R-5543, polyclonal, Fitzgerald) at 1:500 dilution at 4 °C overnight. Peroxidase-labeled anti-rabbit immunoglobulin G secondary antibody and ABC reagent were added using the peroxidase-conjugated avidin ABC kit (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin to visualize histology. Western blot analysis. Total protein was isolated from whole ileum or isolated IECs and separated by SDS-PAGE and analyzed by western blotting as described previously 68 . Mitochondrial and cytosolic extracts were isolated using mitochondrial isolation buffer (70 mM sucrose, 220 nM D-mannitol, 2 mM Hepes), followed by centrifugation at 900×g for 10 min. The supernatant was centrifuged at 10,000×g for 10 min to pellet mitochondria.
Digital image acquisition. Chemiluminescence pictures of western blots were taken using BioRad VersaDoc gel documentation system and quantified by QuantityOne software (Version 4).
GST pulldown assay. Full-length coding region of PHB1 was cloned into the pGEX-6p-1 vector (Addgene) and transformed into BL21(DE3) cells that were then treated with IPTG to induce expression of fusion proteins. pGEX-6p-1 empty vector was used as a negative control. GST fusion proteins (bait protein) were isolated via lysis of bacterial pellets and immobilized onto glutathione agarose beads. 500 μg protein from primary mouse IECs or Mode-K cells (prey protein) was incubated with the GST-protein loaded agarose beads at 4 °C for 2 h. Beads were then pelleted by centrifugation, washed 3 times with GST pull down wash buffer (2 mM HEPES-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Tween), boiled in Laemmli buffer/β-mercaptoethanol, and visualized by SDS-PAGE.

PHB1-GFP deletion constructs.
The generation of pEGFPN1-PHB1 was previously described 59 . Deletion constructs were generated by 2 separate PCR reactions using pEGFPN1-PHB1 full length as template, high fidelity Taq (Invitrogen), and primers shown in Table 1. One primer in each pair for the first PCRs contain a ~ 20 bp complementary region that allows the fusion of the two domain fragments in the second PCR reaction. The product from the second PCR reaction was ligated into pEGFPN1 vector (Clontech) using the Quick Ligation Kit (New England Biolabs) via HindIII and BamHI sites. All deletion constructs were verified by Sanger sequencing.
Luciferase reporter assay. Mode-K cells were co-transfected with pGL3 or pGL3-Nix, siNC or siPHB1, and pRL-CMV (Renilla luciferase as internal control; E2231, Promega). 72 h post-transfection, cells treated with 500 nM rotenone for 2 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (E1910, Promega) and a BioTek luminometer. Relative luciferase was calculated by normalizing firefly luciferase activity to renilla luciferase activity. mitoSOX measurements. Mode-K cells were incubated with Hank's balanced salt solution (HBSS) with Table 1. Primers used for PCR generation of PHB1-GFPN1 deletion constructs. Statistical analysis. Distinct sets of Phb1 iΔIEC and Phb1 fl/fl mice were used for histological analysis (immunofluorescent staining, TEM; n = 8 per group) or biochemical assays (western blot, mtDNA; n = 8 per group). For starvation experiments, measurements were taken from a distinct set of 7 mice per treatment group. For cell culture experiments, measurements were taken from distinct samples. Covariates were not tested. Sample size was calculated as follows: expecting a 97% rate of Phb1 knockout, and setting a twofold increase in Tim50 protein expression as a biologically relevant response to basal Phb1 deletion, with an alpha and beta level set at 0.05 and 0.2, respectively, a sample size of n = 8 mice of each genotype was required (Stata version 9, StataCorp). For western analysis on mouse samples, all samples were included unless protein sample was exhausted on previous blots. For other measurements, no data was excluded. Animals were allocated to experimental groups based on genotype and to assign even numbers of males and females, no further randomization was used.
Investigators were blinded to group allocation during data analysis. All findings for basal alterations in Phb1 deficient mice were replicated in non-starved mice. All cell experiments were performed in at least 2 separate experiments. All data were collected using Microsoft Excel 2018. Values are expressed as mean ± SEM or as individual data points ± SEM. Statistical analysis was performed using unpaired 2-tailed Student's t test for single comparisons, one-way ANOVA with Tukey's post-hoc test for multiple comparisons, or two-way ANOVA with Tukey's post-hoc to test for the interaction of genotype and treatment (starvation or rotenone) (PRISM 8.2, GraphPad Software). P < 0.05 was considered significant.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.